Most researchers studying immunology have been interested rather in adaptive immunity having memory and specificity than in innate immunity, so far. Nevertheless, innate immunity plays an important role in self-defense system of animals. For instance, 1) innate immune cells prohibit the invasion of microorganisms through skin or epitherial cells of the intestines, 2) innate immune cells restrain pathogens invading into blood or body fluid with their phagocytosis, 3) innate immunity preferentially prevents various invading microorganisms from growing in body fluid after infection even before adaptive immunity or phagocytosis is activated since innate immunity does not have specificity. Cells that are responsible for innate immunity use various antimicrobial substances such as simple inorganic compounds (H2O2, NO, etc), antimicrobial peptides and proteins in order to function as the above. Antimicrobial peptides or proteins have been reported to be on mucosal epithelial surface, in body fluid and in intracellular organelles of phagocytes, and to have various sizes, structures and activity (Hancock, R. E. et al., Proc. Natl. Acad. Sci., 2000, 97, 8856-8861). But there are common characteristics, too, that is, most antimicrobial peptides or proteins have complementary positive charge to negative charge of cell membrane of microorganisms, antimicrobial proteins having enzyme activity (proteases or muramidases) hydrolyze the membrane of bacteria and antimicrobial peptides also target in cell membrane of microorganisms (Zhang, L. et al., J. Biol. Chem., 2001, 276, 35714-35722). Owing to these mechanisms, antimicrobial peptides are expected to be very helpful for the development of novel antibiotics that can be effectively used for the bacteria having resistance against conventional antibiotics. Frequent appearance of resistant strains resulted from overuse of chemical synthetic antibiotics evokes the interest in these antimicrobial peptides as well.
Antimicrobial peptides are largely classified two groups: one group is composed of peptides having bipolar α-helical structure and the other group is composed of peptides having β-sheet structure stabilized by intradisulfide bonds. Cysteine containing antimicrobial peptides mostly keep even number of cysteine residues from 2 to 8, which contribute to build intradisulfide bonds, resulting in the completion of a stable structure. Table 1 shows a classification of antimicrobial peptides having β-sheet structure by the number of cysteine residue in a molecule.
TABLE 1β-sheet antimicrobial peptides classified by thenumber of intramolecular systeineCysteinenumberPeptideOriginReference2DodecapeptideRuminants1ThanatinInsects2BombininAmphibian34TachyplesinTachypleus4tridentatusAndroctoninScorpion5ProtegrinPig66α-defensinLeucocytes of7mammalianβ-defensinEpithelial8cells ofmammalian8HepcidinHuman liver91. Romeo, D. et al., J. Biol. Chem., 1988, 263, 9573-9575.2. Fehlbaum, P. et al., Proc, Natl, Acad, Sci., 1996, 93, 1221-1225.3. Goraya, J. et al., Eur. J. Biochem., 2000, 267, 894-900.4. Iwanaga, S. et al., J. Biochem., 1998, 123, 1-15.5. Hetru, C. et al., Biochem. J., 2000, 345, 653-644.6. Ganz, T. et al., Drugs, 2000, 9, 1731-1742.7. Lehrer, R. I. et al., Annu. Rev. Immunol., 1993, 11, 105-128.8. O'Neil, D. A. et al., J. Immunol., 1999, 163, 6718-6724.9. Krause, A. et al., FEBS Lett., 2000, 480, 147-150.
Working mechanism and specificity of antimicrobial peptides depend on the way to work mutually with bacterial cell membranes. Generally, peptides are accepted through self-promoted uptake pathway by working with LPS (lipopolysaccharide) on the surface of Gram-negative bacteria. The first step of the accepting process is that the peptides are adhered to divalent cation-binding sites of LPS on cell surface, and the second step is that the peptides are inserted in cell membrane to form a channel.
In the first step, peptides can bind to LPS with 3 times as high affinity as divalent cations like Mn++ or Mg++, so that they can be substituted for the divalent cations, causing a break down of a general property of cell membrane, especially of outer membrane. Such affected bacterial cell membrane makes a gap temporarily, through which hydrophobic substances, low-molecular proteins or antibiotics can pass and especially peptides are inserted effectively (Piers, K. L. et al., Antimicrob. Agents Chemother., 1994, 38, 2311-2316).
In the second step, peptides are inserted in cell membrane to form a channel, during which magnetism of cation peptides works with anions of bacterial membrane, so that hydrophobic region faces membrane and hydrophilic region faces inner side to form a channel (Hancock, R. E. et al., Adv. Microbial Physiol., 1995, 37, 135-175). The channel is formed well when potential difference is big, the amount of anion lipids is great and the quantity of cholesterol is small. A well-formed channel causes a break down of membrane structure, resulting in the death of bacteria (Falla, T. et al., J. Biol. Chem., 1996, 271, 19298-19303). On the contrary, eukaryotic cells containing a huge amount of cholesterol but a small quantity of anion lipid do not provide a good condition for the working of peptides. Thus, the peptides show a highly selective activity against bacteria. Based on the above reasons, antimicrobial peptides are noticed as novel antibiotics with less cytotoxicity. Besides, the advantages of antimicrobial peptides, as novel antibiotics, are as follows.
1. Preventing the appearance of resistant bacteria by destroying bacterial membrane physically.
2. Working faster than the life cycle of bacteria.
3. Working effectively on resistant bacteria having resistance against conventional antibiotics.
4. Having wide antimicrobial spectrum.
5. Having an anti-endotoxicity effect owing to the binding capacity to LPS, etc.
6. Being able to be mass-produced using genetic engineering techniques and developed as a novel medicine with a less production cost.
From the viewpoint of animal systematic taxonomy, a tunicate belonging to deuterostomia is a kind of invertebrates classified into protochordata with such characteristics as having notochord and dorsal tubular nerve cord during tadpole larva period. Thus, a tunicate can be classified in pre-vertebrata with respect to systematic evolutionistics. Owing to such taxonomical position, a tunicate has been regarded as a model animal to prove evolutional origin of animal immune system. Especially, the body cavity (hemocoel) of a tunicate was observed to have lots of phagocytes having similar forms and functions to granulocytes and macrophages found in circulatory system of vertebrata (Bone, Q., The Origin of Chordates, 1979, 2nd edn). Studies to detect out antimicrobial peptides from body fluid cells of a tunicate have been undergoing and clavanin (Lee, I. H. et al., FEBS Lett., 1997, 400, 158-162; Lee, I. H. et al., Infection and Immunity, 1997, 65, 2898-2903; Zhao, C. et al., FEBS Lett., 1997, 410, 490-492) and styelin (Lee, I. H. et al., Comp. Biochem. Physiol. B Biochem. Mol. Biol., 1997, 118, 515-521; Zhao, C. et al., FEBS Lett., 1997, 412, 144-148) separated from body fluid cells of Styela clava are the representative antimicrobial peptides found out so far.
Two kinds of tunicates inhabit in the country. One is Halocynthia roretzi inhabiting mainly in southwest seashores or raised artificially and the other is Halocynthia aurantium, also called “silky sea squirt”, inhabiting only in Sokcho (Kangwon-Do, Korea) area of the east coast. The former has been studied in Japan many years and disclosed to have an antimicrobial substance in the shape of transformed peptide (tetrapeptide) like halocyamine (Azumi, K. et al., Experientia, 1990, 46, 1066-1068; K. Azumi et al., Biochemistry, 1990, 29, 159-165). But there is no report yet that an antimicrobial peptide is separated from the latter, Halocynthia aurantium. 
Thus, the present inventors investigated if there is any antimicrobial peptide in body fluid cells of Halocynthia aurantium. As a result, the present inventors separated an antimicrobial peptide named as dicinthaurin (Lee, I. H. et al., Biochem. Biophys. Acta, 2001, in press), and further, separated another antimicrobial peptide recently. The present inventors have accomplished this invention by analyzing the structure of the newly separated antimicrobial peptide and confirming the excellent antimicrobial activity thereof.